Diagnostic possibilities of PID in the Republic of Moldova

Departament of Pediatrics, State Medical and Pharmaceutical University “Nicolae Testimiteanu”, Republic of MoldovaAwareness activities: PID are rather rare disorders but they are more common than it was estimated. The rate of recognition and diagnosis of PID is directly dependent on awareness of medical staff (JMF manuscript 2011). According to V.Modell (2011), founder of the JMF, the awareness of medical staff and patients concerning PID remains low all over the world and the majority of patients with recurrent infections are not diagnosed or underdiagnosed. In fact, PID prevalence levels exceed the official data. The handbook entitled “Primary Immunodeficiencies in children” was elaborated for family practitioners, pediatricians and medical residents, and published in 2012. It covers the following topics: general information about children immune system, 10 warning signs for children with PID, clinical presentation, immunological and genetic features of the most common PID syndromes, diagnostic algorithms, and tables with reference values of immunological tests. New diagnostic possibilities of PID in Moldova (2012). Determination of lymphocyte subpopulations using cytoflowmetric analysis, assessment of the IgG subclasses, IgD, evaluation of the C1-estherase activity, phagocytic burst-test using dihydrorhodamine, phagocyitosis killing activity (E.coli) using cytoflowmetric analysis. Algorithm diagnostic of PID in Moldova: Clinical screening – 10 warning signs of PID JMF(clinical features characteristic for well-defined syndromes of PID are also taken into consideration). At risk of PID patients primary are evaluated:: family history, clinical course of disease (especially infectious syndrome characteristic for different forms of PID), documented presence of other features of PID (autoimmunity, malignancy), documented presence of other conditions which can because of infectious susceptibility (structural abnormalities, cystic fi brosis, etc). Children with major risk of IDP are selected and laboratory screening: WBC manual count is made, total IgA, IgG, IgM, Ig E. Patients with major risk of PID are examined using different diagnostic protocols depending on clinical presentation. Different immunological tests are carried out according to the Practice Parameter for the diagnosis and management of PID (Bonilla F. et al., 2005). ESID criteria for PID are used to establish the possible, probable and definitive diagnosis of PID if appropriate. Current situation: 6 children are diagnosed with PID in Moldova currently: • 5 patients with IgA selective deficiency (6-8 years old) • 1 patient with Di George syndrome (4 years old) Currently more than 150 genetic defects determining severe disorders of the immune system have been described. According to the European Society for Primary Immunodefi ciencies database most of the reported immune deficiencies occur with a frequency of not less than 1:100000. The improvement of the diagnosis PID will help to achieve in 2013-2014 the Project “Complex diagnostic approach for patients with rare forms of primary immunodeficiency” which will be achieved with Belarus Republic. Objective of the Project: To elaborate effective diagnostic approach based on the analysis of retrospective and prospective clinical, immunological and genetic data of patients with rare primary immunodeficiency syndromes in the territory of the Republic of Moldova and the Republic of Belarus. It is aimed to perform an in-depth analysis of the disease history, clinical data and immunological disorders in patients with rare (1:100000 to 1:1000000) primary Immunodeficiencies in Moldova and the Republic of Belarus. At least 50 patients with 20 primary immunodefi ciency syndromes of rare incidence will be included. On the basis of the received data in Moldova and the Republic of Belarus a similar algorithm and check-list for clinical and immunological assessment will be developed for children with rare primary immunodefi ciencies. Therefore, a complex diagnostic approach must be elaborated for rare primary immunodefi ciency syndromes, which will include clinical, immunological and genetic features, and will contribute to the same effective as for common immunodefi - ciency syndromes early diagnosis and appropriate treatment. Activities which will help to improve PID diagnosis in Moldova: Implementation of lectures on childhood PID topics in the curricula of postgraduate training for family doctors and pediatricians. PhD research on PID problems. Organization of the J Project meeting in Moldova in the nearest future

Importance of Extranuclear Estrogen Receptor-α and Membrane G Protein–Coupled Estrogen Receptor in Pancreatic Islet Survival

International audienc

Analysis of subcellular metabolite levels of potato tubers (Solanum tuberosum) displaying alterations in cellular or extracellular sucrose metabolism

The expression of a heterologous invertase in potato tubers (Solanum tuberosum) in either the cytosol or apoplast leads to a decrease in total sucrose content and to an increase in glucose. Depending on the targeting of the enzyme different changes in phenotype and metabolism of the tubers occur: the cytosolic invertase expressing tubers show an increase in the glycolytic flux, accumulation of amino acids and organic acids, and the appearance of novel disaccharides; however, these changes are not observed when the enzyme is expressed in the apoplast [Roessner et al. (2001). Plant Cell, 13, 11-29]. The analysis of these lines raised several questions concerning the regulation of compartmentation of metabolites in potato tubers. In the current study we addressed these questions by performing comparative subcellular metabolite profiling. We demonstrate that: (i) hexoses accumulate in the vacuole independently of their site of production, but that the cytosolic invertase expression led to a strong increase in the cytosolic glucose concentration and decrease in cytosolic sucrose, whereas these effects were more moderate in the apoplastic expressors; (ii) three out of four of the novel compounds found in the cytosolic overexpressors accumulate in the same compartment; (iii) despite changes in absolute cellular content the subcellular distribution of amino acids was invariant in the invertase overexpressing tubers. These results are discussed in the context of current models of the compartmentation of primary metabolism in heterotrophic plant tissues

A comprehensive map of molecular drug targets.

The success of mechanism-based drug discovery depends on the definition of the drug target. This definition becomes even more important as we try to link drug response to genetic variation, understand stratified clinical efficacy and safety, rationalize the differences between drugs in the same therapeutic class and predict drug utility in patient subgroups. However, drug targets are often poorly defined in the literature, both for launched drugs and for potential therapeutic agents in discovery and development. Here, we present an updated comprehensive map of molecular targets of approved drugs. We curate a total of 893 human and pathogen-derived biomolecules through which 1,578 US FDA-approved drugs act. These biomolecules include 667 human-genome-derived proteins targeted by drugs for human disease. Analysis of these drug targets indicates the continued dominance of privileged target families across disease areas, but also the growth of novel first-in-class mechanisms, particularly in oncology. We explore the relationships between bioactivity class and clinical success, as well as the presence of orthologues between human and animal models and between pathogen and human genomes. Through the collaboration of three independent teams, we highlight some of the ongoing challenges in accurately defining the targets of molecular therapeutics and present conventions for deconvoluting the complexities of molecular pharmacology and drug efficacy

Overfishing of top predators eroded the resilience of the Black Sea system regardless of the climate and anthropogenic conditions

It is well known that human activities, such as harvesting, have had major direct effects on marine ecosystems. However, it is far less acknowledged that human activities in the surroundings might have important effects on marine systems. There is growing evidence suggesting that major reorganization (i.e., a regime shift) is a common feature in the temporal evolution of a marine system. Here we show, and quantify, the interaction of human activities (nutrient upload) with a favourable climate (run-off) and its contribution to the eutrophication of the Black Sea in the 1980s. Based on virtual analysis of the bottom-up (eutrophication) vs. top-down (trophic cascades) effects, we found that an earlier onset of eutrophication could have counteracted the restructuring of the trophic regulation at the base of the food web that resulted from the depletion of top predators in the 1970s. These enhanced bottom-up effects would, however, not propagate upwards in the food web beyond the zooplankton level. Our simulations identified the removal of apex predators as a key element in terms of loss of resilience that inevitably leads to a reorganization. Once the food web has been truncated, the type and magnitude of interventions on the group replacing the apex predator as the new upper trophic level have no effect in preventing the trophic cascade. By characterizing the tipping point at which increased bottom-up forcing exactly counteracts the top-down cascading effects, our results emphasize the importance of a comprehensive analysis that take into account all structuring forces at play (including those beyond the marine system) at a given time.EUR-OCEANS Integration Project 2007; Marie Curie Intra-European fellowship [FP6-2005-Mobility-5]; Marie Curie European Reintegration Grant [FP7-People- 2009-RG]; EU WETLANET, KNOWSEAS, and MEECE, UK NERC QUEST; US National Science Foundation (DMS-0935617

Identification of a small molecule yeast TORC1 inhibitor with a flow cytometry-based multiplex screen

TOR (target of rapamycin) is a serine/threonine kinase, evolutionarily conserved from yeast to human, which functions as a fundamental controller of cell growth. The moderate clinical benefit of rapamycin in mTOR-based therapy of many cancers favors the development of new TOR inhibitors. Here we report a high throughput flow cytometry multiplexed screen using five GFPtagged yeast clones that represent the readouts of four branches of the TORC1 signaling pathway in budding yeast. Each GFP-tagged clone was differentially color-coded and the GFP signal of each clone was measured simultaneously by flow cytometry, which allows rapid prioritization of compounds that likely act through direct modulation of TORC1 or proximal signaling components. A total of 255 compounds were confirmed in dose-response analysis to alter GFP expression in one or more clones. To validate the concept of the high throughput screen, we have characterized CID 3528206, a small molecule most likely to act on TORC1 as it alters GFP expression in all five GFP clones in an analogous manner to rapamycin. We have shown that CID 3528206 inhibited yeast cell growth, and that CID 3528206 inhibited TORC1 activity both in vitro and in vivo with EC50s of 150 nM and 3.9 μM, respectively. The results of microarray analysis and yeast GFP collection screen further support the notion that CID 3528206 and rapamycin modulate similar cellular pathways. Together, these results indicate that the HTS has identified a potentially useful small molecule for further development of TOR inhibitors

Transcript and metabolite profiling of the adaptive response to mild decreases in oxygen concentration in the roots of arabidopsis plants

Oxygen can fall to low concentrations within plant tissues, either because of environmental factors that decrease the external oxygen concentration or because the movement of oxygen through the plant tissues cannot keep pace with the rate of oxygen consumption. Recent studies document that plants can decrease their oxygen consumption in response to relatively small changes in oxygen concentrations to avoid internal anoxia. The molecular mechanisms underlying this response have not been identified yet. The aim of this study was to use transcript and metabolite profiling to investigate the genomic response of arabidopsis roots to a mild decrease in oxygen concentrations. Arabidopsis seedlings were grown on vertical agar plates at 21, 8, 4 and 1 % (v/v) external oxygen for 0.5, 2 and 48 h. Roots were analysed for changes in transcript levels using Affymetrix whole genome DNA microarrays, and for changes in metabolite levels using routine GC-MS based metabolite profiling. Root extension rates were monitored in parallel to investigate adaptive changes in growth. The results show that root growth was inhibited and transcript and metabolite profiles were significantly altered in response to a moderate decrease in oxygen concentrations. Low oxygen leads to a preferential up-regulation of genes that might be important to trigger adaptive responses in the plant. A small but highly specific set of genes is induced very early in response to a moderate decrease in oxygen concentrations. Genes that were down-regulated mainly encoded proteins involved in energy-consuming processes. In line with this, root extension growth was significantly decreased which will ultimately save ATP and decrease oxygen consumption. This was accompanied by a differential regulation of metabolite levels at short- and long-term incubation at low oxygen. The results show that there are adaptive changes in root extension involving large-scale reprogramming of gene expression and metabolism when oxygen concentration is decreased in a very narrow range

CACHE (Critical Assessment of Computational Hit-finding Experiments): A public–private partnership benchmarking initiative to enable the development of computational methods for hit-finding

One aspirational goal of computational chemistry is to predict potent and drug-like binders for any protein, such that only those that bind are synthesized. In this Roadmap, we describe the launch of Critical Assessment of Computational Hit-finding Experiments (CACHE), a public benchmarking project to compare and improve small-molecule hit-finding algorithms through cycles of prediction and experimental testing. Participants will predict small-molecule binders for new and biologically relevant protein targets representing different prediction scenarios. Predicted compounds will be tested rigorously in an experimental hub, and all predicted binders as well as all experimental screening data, including the chemical structures of experimentally tested compounds, will be made publicly available and not subject to any intellectual property restrictions. The ability of a range of computational approaches to find novel binders will be evaluated, compared and openly published. CACHE will launch three new benchmarking exercises every year. The outcomes will be better prediction methods, new small-molecule binders for target proteins of importance for fundamental biology or drug discovery and a major technological step towards achieving the goal of Target 2035, a global initiative to identify pharmacological probes for all human proteins. [Figure not available: see fulltext.

Complementarity Between a Docking and a High-Throughput Screen in Discovering New Cruzain Inhibitors†

Virtual and high-throughput screens (HTS) should have complementary strengths and weaknesses, but studies that prospectively and comprehensively compare them are rare. We undertook a parallel docking and HTS screen of 197861 compounds against cruzain, a thiol protease target for Chagas disease, looking for reversible, competitive inhibitors. On workup, 99 % of the hits were eliminated as false positives, yielding 146 well-behaved, competitive ligands. These fell into five chemotypes: two were prioritized by scoring among the top 0.1 % of the docking-ranked library, two were prioritized by behavior in the HTS and by clustering, and one chemotype was prioritized by both approaches. Determination of an inhibitor/cruzain crystal structure and comparison of the high-scoring docking hits to experiment illuminated the origins of docking false-negatives and false-positives. Prioritizing molecules that are both predicted by docking and are HTS-active yields well-behaved molecules, relatively unobscured by the false-positives to which both techniques are individually prone